Nucleic acid analogs, including peptide nucleic acids (PNA), internucleotide analogs, and nucleobase analogs, are often studied and characterized by analytical tests and methods at substantially higher concentrations than physiological concentrations under metabolic conditions (Alberts, 1989). Some tests, methods and experiments that may require high concentrations of nucleic acid analogs are intracellular antisense inhibition of transcription and translation of sense DNA and mRNA, labelling of nucleic acid analog reactions, and attaching nucleic acid analogs to surfaces of other heterogeneous media. Analytical methods that may require high concentrations of nucleic acid analogs are gel electrophoresis (Rickwood, 1990), high-performance liquid chromatography (HPLC) (Oliver, 1998), primer extension reactions, polymerase chain reaction (PCR), mass spectroscopy, nuclear magnetic resonance (NMR) and X-ray crystallization.
Nucleic acid analogs with uncharged, neutral backbones, such as peptide-nucleic acids (PNA) (Buchardt, 1992), morpholino-carbamate (Summerton, 1991), methyl phosphonate oligonucleotides (Miller, 1991), and others (Goodnow, 1998) have the advantage of nuclease-resistance for prolonged in vivo stability but are notoriously difficult to solubilize and maintain in aqueous solution, particularly at neutral pH. Such uncharged analogs lack the solubility-facilitating phosphate anions of DNA and RNA. PNA sequences with a N-(2-aminoethyl)-glycine polymer backbone and high purine nucleobase content can be especially difficult to solubilize (Nielsen, 1991). Useful concentrations of PNA sequences with contiguous thymidine nucleobases are also sometimes difficult to achieve. Typically, PNA are dissolved in low pH, primarily aqueous solutions. PNA nucleobases are basic and become protonated and charged at low pH, increasing their solubility under acidic conditions. For the most part, nucleic acid analogs such as PNA are not appreciably soluble in neutral, aqueous conditions. If solution is achieved at neutral pH by elevated temperature, agitation, or other techniques, nucleic acid analogs are prone to precipitation when allowed to stand at or below room temperature. Furthermore, high concentrations of PNA and other nucleic acid analog molecules can aggregate to form insoluble, secondary and tertiary structures that precipitate from solution. These precipitates are often difficult to redissolve.
Aggregation of PNA and other nucleic acid analogs is a significant hindrance to purification, analysis, characterization, measurement, and use of these compounds (Schmidt, 1996). Insolubility and precipitation make the nucleic acid analog concentrations questionable and sample handling and storage unreliable. These limitations are unfortunate because solutions of high concentrations of nucleic acid analogs, e.g. purine-rich PNA are very desirable.
Nucleic acid analogs, such as PNA, are often covalently labelled, or conjugated, with hydrophobic labelling reagents for detection, affinity, or other recognition purposes in labelling reactions, e.g. fluorescent dyes, biotin, digoxigenin. Preferred labelling reaction conditions include aqueous, neutral solutions with high concentrations of the nucleic acid analog and the labelling reagents. Methods or compositions to attain high concentrations of nucleic acid analogs for labelling reactions are desirable.
The labelled nucleic acid analog conjugates that result from labelling reactions are often insoluble in neutral aqueous media. Methods or compositions to attain high concentrations of labelled nucleic acid analog conjugates, e.g. fluorescent dye-PNA, are desirable.
Nucleic acid analog mixtures with a propensity to precipitate, or that demonstrate insolubility, require onerous monitoring of their concentrations for appropriate experimental control. Sometimes dissolution of nucleic acid analogs with basic moieties that become protonated can be achieved at low pH, e.g. 1-5. However, not all tests, methods, experiments, applications or uses for nucleic acid analogs are feasible at acidic pH. For example, nucleic acids may not hybridize with specificity and affinity at acidic pH. In addition, there are several cell biological and molecular applications in which neutral pH is required or preferred (Alberts, 1989).
Certain labels and linkers have been covalently attached to nucleic acid analogs to enhance their solubility in attempts to attain high concentrations for various purposes. For example, hydrophilic moieties such as polyoxyethylene moieties have been conjugated to PNA to improve its solubility (Gildea, 1998). Attaching such solubilizing moieties requires special reagents and extra expense to attain high concentration nucleic acid analog solutions. An additional drawback is the adverse impact on overall synthesis efficiency when attaching solubilizing moieties. Furthermore, such moieties may change the shape, charge, hydrophobicity, and other properties of a nucleic acid analog. Attached moieties may disrupt, prevent, or impair biological utility of the nucleic acid analog function, e.g. hybridization specificity or affinity, antibody/antigen recognition, and enzymatic activity.
Solubility properties of nucleic acid analogs are difficult to predict, and are type and sequence dependent. Therefore, it is desirable to provide solvents and solvent mixtures capable of solubilizing nucleic acid analogs at high concentrations. Conditions which allow long term storage, i.e. one week or more, of nucleic acid analogs such as PNA at .mu.M (10.sup.-6 molar,) to 10 mM (10.sup.-2 molar) concentrations at neutral pH with retention of the PNA in solution are especially desirable.